Malaria is responsible for over a million deaths a year, with most of the victims being young children. One of the most cost-effective interventions to reduce this toll would be the development of a safe and effective vaccine against Plasmodium falciparum, the causative agent of the most severe form of the disease. Individuals living in malaria-endemic regions develop clinical immunity associated with high antibody titers against surface molecules of blood stages (in particular, the merozoite) of the parasite.
Vaccines against the blood stages of the parasite could reduce the morbidity and mortality, particularly among children. They could also accelerate the acquisition of natural immunity, and help maintain it through constant boosting of the immune response by naturally occurring infections.
Passive transfer studies have shown that immunoglobulins from semi-immune individuals can confer clinical immunity to individuals exposed to geographically diverse parasite strains.
Most epitopes recognized by antibodies represent three-dimensional surfaces of an antigen molecule that fit precisely the binding surfaces of the corresponding antibodies (for a review, see Corradin et al., 2007 Endocrine, Metabolic & Immune Disorders—Drug Targets, 7: 259-265).
These epitopes are classified into distinct groups: linear and structural epitopes. Linear epitopes are made of a continuous unstructured stretch of amino acid residues, while structural protein segment(s) which may be discontinuous (for example, loops) or not. Linear epitopes usually have flexible unfolded conformations and are located in large unstructured loops or terminal protein regions. They can be, in general, mimicked by short protein segments obtained either by protein fragment, or, more simply, by peptide synthesis. Thus, at first sight, linear epitopes do not seem to represent a major technical challenge. However, several estimates that only 10% of the antibodies elicited during an immune response are directed against linear epitopes. Thus most antibodies are raised against discontinuous structural epitopes.
Therefore, research has up to now mainly focussed on structural or conformation-dependent epitopes, and on the production of short, structurally stable protein segments, which, as isolated peptides, are able to fold into the native structure and thus be recognized by conformation-dependent antibodies. Such structural or conformation-dependent domains notably encompass globular functional domains (such as zinc-fingers, knottins, animal toxins, FGF molecules, chemokines), and structural motifs of protein made of tandem repeats, such as alpha-helical coiled coil domains. For example, WO 2007/020520 in the names of Université de Lausanne and Institut Pasteur describes Plasmodium peptidic antigens, such as the P27 antigen (SEQ ID NO: 27 in this PCT international application), which mimic the alpha-coiled coil domain of the native MAL6P1.37 (also referred to as PFF0165c) protein of Plasmodium falciparum 3D7.
By contrast, the natively unfolded or unstructured regions of Plasmodium proteins (which are also referred to as IUPs, standing for Intrinsically Unstructured Proteins, see Zhi-Ping Feng et al., 2006 Molecular & Biochemical Parasitology, 150: 256-267) do not fold in any particular unique structure. Natively unfolded or unstructured regions of Plasmodium proteins can be identified in the sequence of proteins by bioinformatics analysis (Oldfield et al. 2005 Biochemistry 44: 1989-2000; Linding et al. 2003 Structure (Camb) 11: 1453-1459; Coeytaux and Poupon 2005 Bioinformatics 21: 1891-1900; Dosztanyi et al. 2005 Bioinformatics 21: 3433-3434). At least 15 online services have been established to identify such structures. One of the most widely used services is DisEMBL large-scale sequence analysis (Linding et al. 2003 Structure (Camb) 11: 1453-1459; Zhi-Ping Feng et al., 2006 Molecular & Biochemical Parasitology, 150: 256-267). The DisEMBL comprise three different predictors, Loops/coils, Hot-Loops and REMARK465, which are based on the same algorithm but different training sets, to predict the ordered or disordered state of a residue (cf. dis.embl.de/html/help.html).
In malaria parasites, about 40% of genome-encoded proteins contain natively unstructured regions with segments longer than 50 amino acids. Many of these proteins have a highly hydrophilic amino acid sequence that cannot form a hydrophobic core needed to stabilize a globular structure. Some of these proteins or fragments thereof are currently being developed as vaccine candidates. These comprise the repeat region of the circumsporozoïte (CS) protein, as well as selected segments of MSP2, MSPS and GLURP of P. falciparum, and the N-terminal and repeat regions of the CS protein of P. vivax. 
One of the difficulties in developing peptide antigen from such natively unfolded or unstructured regions of Plasmodium proteins is the difficulty of selecting candidates from the large number of predicted unstructured regions found in genomes, and their potential amyloidogenicity.
The inventors have identified polypeptides deriving from a protein of Plasmodium falciparum, which are unfolded or unstructured polypeptides, and which show improved properties with respect to prior art peptides or polypeptides.
The P. falciparum protein, from which the polypeptides of the invention derive, is the MAL6P1.37 (also referred to as PFF0165c) protein of Plasmodium falciparum 3D7 (accession number of the protein sequence: XP—966024). This 1103 amino acid-long protein is encoded by chromosome 6 of P. falciparum and only is a predicted protein, with no known function yet.
The present inventors demonstrate that unfolded or unstructured polypeptides, which derive from this predicted protein, have a high antigenicity, a high immunogenicity, and have a parasite-killing activity in the Antibody-Dependent Cellular Inhibition (ADCI) assay that is as high as, or higher than the structured P27 peptide disclosed in WO 2007/020520.
Furthermore, the total proportion of individuals who, under natural exposure to a malaria parasite, respond by specific IgG1 and IgG3, i.e., the most critical IgG subclasses, is higher for the polypeptides of the invention, such as the P27A than for other antigens, including peptide P27.
For an illustration of this effect and advantage, please see e.g., the prevalence values indicated in Table 2 in example 2 below, as well as the prevalence values indicated below the diagram of FIG. 4. The total proportion of individuals who, under natural exposure to a malaria parasite, respond by specific IgG1 and IgG3, is higher for the polypeptide of the invention P27A (anti-P27A specific IgG1: 86.7% of the individuals; anti-P27A specific IgG3: 82.2% of the individuals) than for other antigens, including peptide P27 (anti-P27 specific IgG1: 6.7%; anti-P27 specific IgG3: 95.6%).
Moreover, the polypeptides of the invention are strongly associated with clinical protection against malaria. In human beings under natural exposure to the parasite, the polypeptides of the invention induce specific antibodies (IgG1 and IgG3) that are very strongly associated with a state of resistance to malaria (statistical association, as assessed by a multivariate analysis made in accordance with the methodology described in Roussilhon et al. 2007 PLoS medicine 4(11): 1791-1803, with p<0.0014). Parasite-induced antibodies that are specific of the polypeptides of the invention are present in individuals, who resist to malaria and are absent, or are present at low titers, in individuals, who have malaria attack.
This association with resistance to malaria is much stronger for the polypeptides of the invention than for the prior art antigens, such as the P27 peptide disclosed in WO 2007/020520. Additionally, the polypeptides of the invention, in particular the conservative variant as defined herein, are different from those disclosed in this prior art.
Although there has been some progress in the treatment of malaria, the development of a safe and effective malaria vaccine remains an urgent unmet medical need for vast populations living in malaria-endemic region.
This object has been achieved by providing a polypeptide which is a fragment or variant of PFF0165c protein of P. falciparum, the amino acid sequence of which is i) the sequence of SEQ ID NO: 6; ii) the sequence of a fragment of said sequence of SEQ ID NO: 6; or ii) a conservative variant, which derives from said sequence of SEQ ID NO: 6 or from said fragment of SEQ ID NO: 6 by at least one conservative amino acid substitution and/or at least one conservative internal amino acid deletion.
For an illustration of this effect and advantage, please see e.g., table 3 in example 2 below, showing the F and p values of a multivariate analysis of the association with a state of resistance to malaria, wherein antibodies specific of the P27A polypeptide of the invention have the highest F ratio (28.55, with p<0.0001).